Botulinum neurotoxins (BoNTs) are a group of homologous protein toxins, produced by various strains of Clostridium botulinum and in some cases C. butyricum and C. baratii (Schiavo et al., Physiol. Rev. 80:717-766, 2000). BoNTs elicit the characteristic flaccid paralysis of botulism by blocking acetylcholine release at the neuromuscular junction, through the cleavage of proteins involved in exocytosis. The seven serotypes of BoNTs (A-G) are synthesized and released by the clostridia as inactive ˜150 kDa protein precursors (Sakaguchi Pharmacol. Ther. 19:165-194, 1983; Minton, Curr. Top. Microbiol. Immunol. 195:161-194, 1995; Oguma et al., Microbiol. Immunol. 39:161-168, 1995; Lacy et al., J. Mol. Biol. 291:1091-1104, 1999; Popoff et al., Structural and genomic features of clostridial neurotoxins, in: J. E. Alouf, J. H. Freer (Eds.) Comprehensive Sourcebook of Bacterial Protein Toxins, Academic, London, 1999). The BoNTs are activated by proteolytic cleavage to generate disulfide-linked di-chain toxins (Sakaguchi, supra, 1983; Minton, supra, 1995; Oguma et al., supra, 1995; Lacy et al., supra, 1999; Popoff et al., supra, 1999), which are amongst the most potent biological poisons known with a mouse lethal dose (LD50) of 0.1-1 pg BoNT/g.
The molecular architecture of the BoNTs is conserved and related to their mode of neural intoxication. Heavy chain (HC, ˜100 kDa) consists of a C-terminal 50 kDa domain (HCC) involved in specific binding to the pre-synaptic membrane via gangliosides and a protein co-receptor (Dong et al., J. Cell Biol. 162:1293-1303, 2003). The N-terminal 50 kDa domain of HC (HCN) is involved in the subsequent translocation of the Light chain (LC, 50 kDa) into the cytosol (Schiavo et al., supra, 2000; Sakaguchi, supra, 1983; Minton, supra, 1995; Oguma et al., supra, 1995; B. D. Lacy et al., supra, 1999; Popoff et al., supra. 1999).
BoNT LCs are zinc metalloproteases that cleave one of three proteins, collectively termed SNARE proteins, which are core components of the machinery that mediates small synaptic vesicle (SSV) fusion, which is responsible for the release of neurotransmitters from nerve terminals. BoNT/A and BoNT/E cleave SNAP-25, BoNT/C1 cleaves syntaxin and SNAP-25, while BoNT/B, /D, /F and /G cleave the vesicle associated membrane protein (VAMP)/synaptobrevin, an integral membrane protein of SSV (Schiavo et al., supra, 2000).
Thus, the BoNTs display exquisite substrate specificity and recognize structurally distinct substrates. This unique substrate specificity may be a model to study substrate recognition by bacterial toxins. However, studies utilizing the holotoxin are constrained by a number of issues, including the intrinsic toxicity of the holotoxin, the lack of tools for genetic manipulation of the clostridia, and the need to activate the holotoxin, a source of inherent error in the analysis of catalytic activity. Studies of other bacterial toxins, such as diphtheria toxin, have overcome these difficulties through the generation of non-toxic catalytic derivatives (Collier, Toxicon. 39:1793-1803, 2001). Similarly, the generation of recombinant, catalytically active LC will allow more detailed structure-function studies of BoNTs.
LC has been expressed as a recombinant protein in E. coli, with varied success. Early attempts to expressed LC in E. coli often resulted in limited expression and poor solubility at concentrations >1 mg/ml (Li et al., Biochemistry 33:7014-7020, 1994; LaPenotiere et al., Toxicon. 33:1383-1386, 1995; Zhou et al., Biochemistry 34:15175-15181, 1995; Kadkhodayan et al., Protein Express. Purif. 19:125-130, 2000). The limited solubility of LC purified from the BoNT, suggests that solubility is an intrinsic property of the LC. Recently, Li and Singh (Li et al., Protein Express. Purif. 17:339-344, 1999) reported the good expression and purification of recombinant LC, which has been used for kinetic and spectroscopic characterization of toxin action.